Fig. 2: Misoprostol prevents hypoxia-induced mitochondrial perturbations in primary neonatal cardiomyocytes.

A Primary ventricular neonatal cardiomyocytes (PVNCs) treated with 10 μM misoprostol (Miso) with or without exposure to 1% O₂ (HPX) for 24 h. MitoView Green was included in all conditions to show mitochondrial morphology. Cells were stained with hoechst (blue) and imaged by standard fluorescence microscopy. B Quantification of cells in (A), where the number of cells with elongated and fragmented mitochondria are expressed as a percentage of all transfected cells in 30 random fields, across 3 independent experiments. C Quantification of PVNC’s treated as in (A). Cells were stained with TMRM (red) and hoechst (blue) and imaged by standard fluorescence microscopy. Red fluorescent signal was normalized to cell area and quantified in 30 random fields, across 3 independent experiments. D Human induced pluripotent stem cell-derived cardiomyocytes (H-iPSC-CMs) treated as in (A). Cells were stained with TMRM (red) and hoechst (blue) and imaged by standard fluorescence microscopy. E Quantification of cells in (D), as in (C). F PVNC’s treated as in (A). Cells were stained with MitoSOX (Red) and hoechst (blue) and imaged by standard fluorescence microscopy. G Quantification of cells in (F), as in (C). H Quantification of PVNC’s treated as in (A). Cells were stained with dihydrorhod-2AM (Red) and hoechst (blue) and imaged by standard fluorescence microscopy and quantified as in (C). I H-iPSC-CMs treated as in (A). Cells were stained with dihyrorhod-2AM (Red) and hoechst (blue) and imaged by standard fluorescence microscopy. J Quantification of cells in (I), as in (C). K Quantification of PVNC’s treated as in (A). Cells were stained with hoechst (blue) and calcein-AM quenched by cobalt chloride (CoCl₂, 5 μM) to assess permeability transition, where green fluorescent signal was normalized to cell area and quantified in 30 random fields, across 3 independent experiments. L H9c2 cells treated as in (A), GW-1-Mito-pHred (red) was included in all conditions to visualize mitophagy. Cells were stained with hoechst (blue) and imaged by standard fluorescence microscopy. M Quantification of cells in (L), where red fluorescent signal was normalized to cell area and quantified in 15 random fields, across 3 independent experiments. N Calculated oxygen consumption rates (OCR) determined by Seahorse XF-24 analysis to evaluate mitochondrial function. O Quantification of PVNC’s treated as in (A). Live cells were stained with calcein-AM (green), and necrotic cells were stained with ethidium homodimer-1 (red). Percent (%) dead was calculated across 30 random fields, across 3 independent experiments. P Quantification of PVNC’s treated as in (A). Cells were fixed, stained with hoechst (blue), and immunofluorescence was performed using a HMGB1 primary antibody (green). Cells were then imaged by standard fluorescence microscopy. Green fluorescent signal was then normalized to nuclear area and quantified in 30 random fields, across 3 independent experiments. All data are represented as mean ± S.E.M. *P < 0.05 compared with control, while **P < 0.05 compared with hypoxia treatment, determined by 1-way ANOVA or 2-way ANOVA where appropriate. Three * indicates P < 0.05 compared to both control and hypoxia treatment.